Peptide, immunogenic composition and vaccine or medical preparation, a method to immunize animals against the hormone LHRH, and analogs of the LHRH tandem repeat peptide and their use as vaccine

ABSTRACT

A peptide that comprises a modified tandem GnRH decapeptide sequence which allows for a testosterone level that is essentially nondetectable after vaccination with the peptide in a suitable dosage and/or allows for an immunogenic response that allows for the effective discrimination between GnRH-I and GnRH-II and a method for the immunocastration of pigs.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of co-pending application Ser.No. 09/659,983, filed Sep. 12, 2000, now U.S. Pat. No. ______, which isa continuation-in-part of application Ser. No. 09/274,048, filed Mar.22, 1999, now U.S. Pat. No. 6,284,733, issued Sep. 4, 2001, which is acontinuation of application Ser. No. 08/981,557, filed Dec. 5, 1997, nowU.S. Pat. No. 5,885,966, issued Mar. 23, 1999 as a national entryapplication under PCT/NL96/00223 filed Jun. 6, 1996, which claimspriority under U.S. application Ser. No. 08/477,298 filed Jun. 7, 1995and U.S. application Ser. No. 08/476,013 filed Jun. 7, 1995. Thecontents of all of these references being incorporated by this referencein their entirety.

TECHNICAL FIELD

[0002] This invention relates to a peptide suitable for eliciting animmune response against forms of Gonadotropin Releasing Hormone (GnRH)also referred to as Luteinizing Hormone Releasing Hormone (LHRH). Theinvention further relates to immunogenic compositions and vaccines,pharmaceuticals, and other medicinal preparations based on such apeptide. The invention further relates to the use of such a vaccine ormedicinal preparation in a method of immunizing a mammal against GnRH toinfluence reproductive or behavioral characteristics of that mammal andin a method of improving the carcass quality of pigs. The invention alsorelates to a peptide suitable for eliciting a selective immunogenicresponse against GnRH-I or GnRH-II. Further, the invention relates toantibodies against GnRH-I, to compositions comprising these antibodiesand to the use of the peptides in pharmaceutical compositions or in thepreparation for a medicament for the treatment of prostate cancer.

BACKGROUND

[0003] GnRH-I (in the literature generally referred to as “GnRH”) is asmall 10 amino acid long peptide (decapeptide) from the hypothalamus.The amino acid sequence of GnRH-I (SEQ ID NO: 1) can be represented bythe following three-letter code:

[0004] pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂

[0005] or the corresponding one letter code where pE is pyroglutamicacid and # is amide:

[0006] pE H W S Y G L R P G#.

[0007] GnRH-I acts at the hypophysis to cause an increase in release ofbiologically active Follicle-Stimulating Hormone (FSH) and LuteinizingHormone (LH) in the blood, which in turn stimulate the development ofthe testes in the growing male animal and the synthesis of malesteroids. In the growing female animal the development of the ovaries isstimulated, as is the development of follicles within the ovary,synthesis of female steroids, and ovulation.

[0008] It is known that GnRH-I, if coupled to a carrier protein, can beused to vaccinate animals. Such a vaccination can be carried out forvarious reasons, all of which are connected with the natural function ofthe GnRH-I. As is known, a drastic reduction of LH and/or FSH in theblood inhibits the production of male steroids or androgens and sperm inthe testis of the male and the formation of female steroids orprogestogens and estrogens and follicle maturation in the ovary of thefemale. Such a reduction in the amounts of androgens, progestogens andestrogens in the blood, to a level comparable to that obtainable byremoving the testes or ovaries via castration, can be achieved byeffective immunization of the animal against GnRH-I. In male animals, inmany cases the testes then appear to develop slowly or not at all, withno synthesis of androgens (male steroid hormones) and no formation ofspermatozoa. In female animals the activity of the ovaria appears todiminish, with no synthesis of estrogens and progestogens (femalesteroid hormones), and inhibition of ripening of follicles andovulation.

[0009] Recently it was reported that a second form of GnRH (GnRH-II) ispresent in primate brain (Lescheid et al., Endocrinol. 138 (1997)5618-5629) and a gene for this second GnRH molecule was cloned from ahuman genomic library (GnRH-II, (SEQ ID NO: 2) (White et al., PNAS USA95 (1998) 305-309). Mammalian GnRH-I (SEQ ID NO: 1) is hardly expressedoutside the brain. A few exceptions are known in this respect. GnRH I ispresent in the endometrium of women with a menstrual cycle (Casan et al.Fertil. Sateril. 1998, 70, 102-106) and is expressed during pregnancy inthe human placenta (Kelly et al., DNA cell Biol. 1991, 10, 411-421).GnRH mRNA was found in ovary, testis, thymus, placenta and hypothalamusof the rat (Oikawa et al., Endocrinology, 1990, 127, 2350-2356).Expression of GnRH was detected in immune tissue (spleen, thymus andlymphocytes) of pigs (Weesner et al., Life Sci, 1997, 61, 1643-1649).

[0010] GnRH-II is expressed in many tissues outside the brain, and isfound in especially high concentrations in the kidneys, bone marrow andprostate. The presence of GnRH-II in diverse tissues other than thebrain suggests that GnRH-II may have multiple functions. In addition,the strictly conserved structure of the GnRH-II peptide throughoutdiverse vertebrate species suggests that this neuropeptide possessesvital bioactivities. Until now, however, the functions of GnRH-II havebeen practically unknown. Several types of differentiated lymphocytes,such as T- and B-lymphocytes and mast cells, produce GnRH and GnRH-likepeptides. Significant numbers of the latter cell type are present in thekidney, bone marrow and prostate, perhaps contributing to the highGnRH-II expression in these tissues. GnRH II seems less involved inreproduction as compared to GnRH-I. In the hypogonadal mouse, mousewhich lacks the GnRH-I gene, GnRH-II producing cells are present in thesame distribution as in a normal mouse, but this is not sufficient tocause normal gonadal development in these mice (Chen et al., FEBSLetters 435 (1998) 199-203). However, macaques in luteal phase of themenstrual cycle showed a marked increase in plasma luteinizing hormoneconcentrations after intravenous administration of GnRH-II, but thisincrease could not be induced during the mid follicular phase (Lescheidet al., Endocrinol. 138 (1997) 5618-5629).

SUMMARY OF THE INVENTION

[0011] The invention now provides the insight that by providing peptidesequences that allow discrimination between the different types of GnRH,more adequate and efficient use can be made of the variation ordifference in immunological response to the different types of GnRH.More particularly, the invention provides the insight that improvementsin the efficacy and selectivity of the vaccines against GnRH-I can beachieved.

[0012] Immunization against GnRH-I is effective in neutralizing GnRH-Iand results in reduced gonadotropin levels and blocking of gonadalsteroid synthesis. However, nothing is known about any physiologicaleffects of the antibodies raised against GnRH-I on the function ofGnRH-II. As GnRH-II is mainly synthesized and secreted in the kidneys,antibodies raised against GnRH-I that cross-react with GnRH-II mayaffect kidney function. To obviate possible side effects of GnRH-Iimmunization on kidney function, it would be desirable to direct theantigenic response of an immunocastration vaccine specifically towardGnRH-I and to avoid possible harmful side-effects due to neutralizationof non-gonadal GnRH-II.

[0013] If the reproductive capacity alone, often with its accompanyingsexual behavior, of a species needs to be annulled, it would bepreferred to aim at an immunocastration vaccine specificallyneutralizing GnRH-I. Hence the desire to come to a selectiveimmunization against Gonadotropin Releasing Hormone(s), preferablyselective against GnRH-I.

[0014] In veterinary medicine, 100% effective immunization againstGnRH-I could be used for the sterilization of, e.g., small domesticanimals such as male and female cats and dogs, or for the treatment ofaggressiveness in male dogs and bulls, simply by vaccination instead ofby drastic surgery such as castration or ovariectomy. Other conceivablereasons for immunization against GnRH-I are to prevent heat in femaleanimals, such as dogs, cats and cows, and restlessness in male animalsbeing fattened for slaughter.

[0015] In human health care, immunization against GnRH-I and/or GnRH-IIcan be used in the treatment of prostate cancer and breast cancer and,if required, in the treatment of some forms of pituitary carcinoma. Inthe case of prostate cancer, it might be more desirable to neutralizeboth GnRH-I and GnRH-II, as the latter isoform is also highly expressedin prostate tissue.

[0016] Another use of a vaccine against GnRH-I is in the field of stockbreeding, particularly the fattening of pigs for slaughter. The meat ofmale, sexually mature pigs (boars) has a typical odor, the so-called“boar taint” or “boar odor”. In the testes of the sexually mature pig,many C19-delta-16 steroids are formed which are stored in the fat tissueof the animal (Patterson, J. Sci. Food Agric. 19, 31-38 (1968); Brooksen Pearson, J. Anim. Sci. 62, 632-645 (1986); Claus, Zeitschrift.Tierzüchtg. Züchtungsbiol. 93, 38-47 (1976); Claus, Acta Endocrinol.(Copenh.) 91, Suppl. 225, 432-433 (1979)). These steroids are mainlyresponsible for the formation of the disagreeable urine-like odor whenthe meat is heated (Fuchs, Swedish J. Agric. Res. 1, 233-237 (1971);Bonneau, Livest. Prod. Sci. 9, 687-705 (1982)). Owing to this unpleasantodor, meat of male sexually mature pigs is generally unsuitable forconsumption and unfit for export. Because about 10% of the maleslaughter pigs are already sexually mature before the slaughter time,this potentially entails a great loss for the pig farming industry.

[0017] In order to control and prevent these losses, nearly all malepiglets are castrated when they are young with a surgical procedure thatis generally executed without any form of anesthesia. Apart from theanimal-unfriendly aspect of such a castration, castration also leads toinfections, growth inhibition, and a final carcass quality inferior tothat of an intact animal, at least as long as that intact animal has notyet developed boar taint (Walstra, Livest. Prod. Sci. 1, 187-96 (1974)).

[0018] An animal-friendly alternative consists in the reduction of theGnRH-I concentration in the pig pituitary by means of immunizationagainst GnRH-I, the so-called immunocastration. This reduction in GnRH-Ilevels leads to a reduction in the concentrations of biologically activeFSH and LH, which in turn will inhibit development of the testes in thegrowing animals and inhibit the synthesis of testicular steroids,including androstenone, testosterone and estrogens. This method preventsthe occurrence of boar taint in male pigs at slaughter time and makessurgical castration unnecessary as androstenone levels are reduced tolow or undetectable levels (Oonk et al., 1995, Livestock productionScience 42, 63-71).

[0019] A strict requirement for an acceptable vaccine against boar taintis that in almost all pigs development of the testes is delayed to suchan extent that boar taint will not have occurred at the time ofslaughter, and that in the case that the vaccine does not reduce testisdevelopment in an animal, this can be easily detected in a too largetestis size in comparison to successfully immunocastrated pigs.

[0020] In the existing literature and previous patent applicationsregarding the anti-fertility properties of vaccines against GnRH-I, theresults of vaccinations often appear to be variable. In most of thedescribed studies, either a small percentage of the vaccinated animalsdo not respond to the vaccination, or large doses of vaccine, multiplevaccinations or commercially unacceptable adjuvants are needed toproduce the desired effect (Hoskinson et al., 1990, Austr. J. Biotech.4, 166-170; Falvo et al., (1986) J. Anim. Sci. 63:986-994; Clarke etal., 1998, Endocrinology 139, 2007-2014; Adams T. E. and B. M. Adams,Feedlot performance of steers and bulls active immunized againstGonadotropin-Releasing Hormone, J. Anim. Sci. 1992, 70: 1691-1698; Brownet al., Immunization of sheep against GnRH-I early in life: effects ofreproductive function and hormones in rams, Journal of reproduction andFertility (1994) 101, 15-21; Ferro et al., Immunological castrationusing a Gonadotropin-releasing Hormone analogue conjugated to PPD, Foodand agricultural immunology, 1995, 7, 259-272; U.S. Pat. No. 4,608,251;Int. Patent Appl. WO 88/05308).

[0021] Some studies suggested an efficacy of 100% of a vaccine againstGnRH-I, but the vaccine was not tested in a large number of animals(Ladd et al. (1994), Development of an antifertility vaccine for petsbased on active immunization against Luteinizing Hormone releasinghormone, Biology of Reproduction 51, 1076-1083; J. G. Manns and S. R.Robbins (1997). Prevention of boar taint with a recombinant based GnRHvaccine, In: Boar taint in entire male pigs, Proceedings of a meeting ofthe EAAP working group “Production and Utilization of Meat from EntireMale Pigs,” EAAP Publication No. 92, 137-140;); other studies report theefficacy of the vaccine as the mean value of the treated animals, sinceindividual values did not show a clear difference between immunized anduntreated controls (Bonneau et al., J. Anim. Sci. 72, 14-20 (1994);Hennesy et al., 1997. Elimination of boar taint: a commercial boar taintvaccine for male pigs. In: Bonneau, M., Lundström, K. and Malmfors, B.(Eds.), Boar taint in entire male pigs. Wageningen Pers, Wageningen,EAAP Publication No. 92, 141-145).

[0022] The difficulty in preparing this type of vaccine probably iscaused by the phenomenon of tolerance. Self substances such as hormonesare not recognized as foreign but rather are tolerated by the immunesystem. Normally, no antibodies are elicited against self-substances. Inorder for a vaccine to be successful, it must be sufficiently foreign.Only when the vaccine is foreign enough will the immune system nottolerate the vaccine and the production of antibodies be induced.Conversely, however, the antibodies must still be capable of recognizingthe hormone, and thus the vaccine cannot be too “foreign.”

[0023] As these conditions appeared to be mutually exclusive, it was notcertain, until recently, if such substances could be prepared at all.One attempt to produce GnRH-like peptide vaccines consisted of thereplacement of Gly at position 6 of the GnRH-I decapeptide by adextrorotary amino acid (D-Tryp; Chaffaux et al., Recueil de MédicineVétérinaire 161 (2), 133-145, 1985). It was, however, demonstrated thata vaccine preparation containing this modified GnRH-peptide performedeven less well than the normal GnRH-I decapeptide (European PatentApplication-464,124 A).

[0024] Recently, we have shown definitively that it is possible toelicit an effective antibody response in all individuals vaccinatedagainst GnRH-I (Meloen et al., Vaccine 12, 741-746 (1994)). In theseexperiments pigs were vaccinated twice with a GnRH-I vaccine thatdeparts from the classical type of GnRH-I vaccine (GnRH-I coupled to acarrier protein, in Freund's adjuvant), namely the tandem-GnRH-I vaccine(European Patent No. 0464124). In this publication a peptide isdescribed which is characterized in that it comprises at least 2 GnRH-Isequences in tandem (SEQ ID NO: 3) according to the general formula

Z¹-Glx-His-Trp¹-Ser-Tyr-Gly-Leu-Arg-Pro[-Gly-X-Gln-His-Trp²-Ser-Tyr-Gly-Leu-Arg-Pro]_(n)-Gly-Z²,

[0025] in which amino acids are designated according to the three-lettercode, Trp¹ and Trp² are tryptophan (Trp) or formylated tryptophan(N(indole)-formyl-tryptophan), n is a number having a value of at least1, X is either a direct bond or a spacer group between the amino acidsGly and Gln, Z¹-Glx is either pGlu (pyro-glutamic acid) or Gln havingattached thereto a tail comprising one or more additional amino acids,and Gly-Z² is either Gly-NH₂ or Gly having attached thereto a tailcomprising one or more additional amino acids. In this general formula,X may be a direct bond between the amino acids glycine and glutamine,i.e., these amino acids are interconnected directly without anintermediate link (via the normal peptide bond). The tandem-GnRH-Ivaccine invention also comprises peptides in which the GnRH-I sequencesare interconnected via spacers. The nature of the spacer group may varygreatly, from one or more amino acids to a shorter or longer hydrocarbonchain and other compound groups or molecules. In the above generalformula, Z¹-Glx preferably stands for pGlu (pyro-glutamic acid), but canalso stand for Gln having attached thereto a tail comprising one or moreadditional amino acids, e.g., to be used for coupling of the peptide toa carrier protein. In the above general formula, Gly-Z² stands for, forexample, Gly-NH₂, or Gly having attached thereto a tail comprising oneor more additional amino acids, e.g., to be used for coupling of thepeptide to a carrier protein. Preferably, Gly-Z² stands for Gly-Cys-NH₂,the C terminal cysteine being added in connection with a possiblecoupling of the peptide to a carrier protein.

[0026] From PCT International Patent Publication WO 96/40755, it isknown that the tandem-dimer principle applied to a variant of the GnRH-Imolecule resulted in a vaccine that was highly effective in several mildadjuvants, namely Specol and a double oil emulsion, and was alsoeffective in low doses. In this case, the variant of the GnRH-I moleculewas formed by substitution of the sixth amino acid Gly of thedecapeptide by a dextrorotatory (D-) amino acid, D-Lys, following whichthe resulting peptide was dimerized and coupled to a common carriercompound, ovalbumin. Thus, whereas a vaccine using D-amino acidsubstitutions of Gly at position 6 of the original and a single GnRH-Idecapeptide with a D-amino acid decreased the immunogenicity as comparedto the original GnRH-I sequence (Chaffaux et al., Recueil de MédicineVétérinaire 161 (2), 133-145, 1985), such substitutions with a D-aminoacid applied to a tandem-dimer GnRH-I vaccine were able to generate evenmore immunogenic GnRH-I vaccine preparations. Nevertheless, the methodfor vaccination required a repeat dosage of the vaccine in order to becompletely effective. The necessity of an additional booster dosage inorder to achieve essentially 100% vaccination of mammals against GnRH-Iis a disadvantage of the known peptides. We also found that, in certaincases, use of the (D-Lys⁶)GnRH-I tandem dimer (i.e., the GnRH-I tandemdimer, with and without the D-Lys⁶ replacement) resulted in very low butstill measurable amounts of testosterone, which is undesirable and adisadvantage of the (D-Lys⁶)GnRH-I tandem dimer.

[0027] The present invention provides peptide sequences that providealternatives to the tandem D-Lys⁶ GnRH-I when applied in vaccines thatresult in vaccines that are effective for immunocastration.

[0028] An aspect of the present invention is the determination of theextent to which the amino acids in the tandem GnRH-I sequence can bevaried while the resulting substituted tandem GnRH-I is still able toproduce an immunogenic response to GnRH-I sufficient forimmunocastration. Thus, the invention provides for the generation of apeptide sequence that can induce the production of antibodies againstGnRH-I, which are also sufficiently competitive, both in amount andactivity.

[0029] A further aspect of the present invention is to provide a peptidesequence that selectively induces the production of antibodies againstGnRH-I, while inducing little or no immune response towards GnRH-II. Apreferred embodiment thereof is a peptide sequence that not onlyselectively induces the production of antibodies against GnRH-I but isalso effective in immunocastration, while an immune response to GnRH-IIis reduced or absent.

[0030] The present inventors have found that in the tandem GnRH-Ipeptide sequence, various amino acids can be replaced, resulting in adecreasing resemblance to the self-hormone while at the same timeretaining or even increasing the ability of the peptide to elicit GnRH-Ibinding antibodies. Also, certain replacements of amino acids in theGnRH-I peptide sequence result in a selective immune response towardsGnRH-I and to a reduced or absent immune response towards GnRH-II.

[0031] In an aspect of the invention, certain modified tandem GnRH-Ipeptide sequences provide for vaccines that are not only capable ofreducing testes growth in male animals but are also capable ofessentially reducing testosterone levels to a degree that can not bedetermined by conventional techniques.

[0032] Further, vaccines prepared from these peptide sequences expressan activity that in most cases eliminates the need for the secondbooster immunization, as with the conventional D-Lys⁶ tandem GnRH-I, toachieve essentially 100% activity. An activity or efficacy of 100% inthe terms of the present invention is defined as a testosterone levelthat is essentially undetectable with conventional techniques after asingle vaccination.

[0033] One of the most notable features of the present invention is thatantibodies raised by these alternative GnRH vaccines discriminatebetween GnRH-I and GnRH-II. Thus peptides according to the inventionexpress an increased or retained activity against GnRH-I, while at thesame time a reduced or absent immune response to GnRH-II are found. Thisallows for the development of peptides that express an inverse effect;that is, they express an increased or retained activity against GnRH-IIwhile at the same time expressing a reduced or absent immune responseagainst GnRH-I.

[0034] The invention relates in one aspect to a peptide that comprises amodified tandem GnRH-I decapeptide sequence whereby vaccination with thepeptide in a suitable dosage allows for a testosterone level that isessentially non-detectable.

[0035] The invention also relates to a peptide that comprises at leasttwo coupled GnRH-I decapeptide sequences, optionally coupled through aspacer, which allows for an immunogenic response that allows for theeffective discrimination between GnRH-I and GnRH-II.

[0036] The peptides according to the invention are sufficiently like thehormone but at the same more “foreign” to the immune system and have anincreased capability to induce the production of antibodies directedagainst the hormone.

[0037] A feature of the invention is that individual tandem units can bedimerized to further enhance its immunogenicity without losing thepossibility of coupling the peptide or peptide composition to a carriercompound protein.

[0038] The techniques for dimerization and coupling of the tandem to acarrier similar to those described in PCT International PatentPublication WO 96/40755 may be used. It is also envisioned that peptidescontaining only a portion of the GnRH-I or II peptide sequences can beused in the present invention. Examples thereof are nonapeptides andundecapeptides.

[0039] The linkers for use in the peptides according to the inventioncan be selected from the linkers described elsewhere in this applicationor linkers such as

[0040] Linkers are used for coupling two or more dimerized peptidesequences. The amino acid which is used to replace the amino acid in thetandem peptide sequences is preferably an amino acid which is a relativesimple one, such as alanine. In a preferred embodiment therefor, thedifferent amino acid is alanine. Other amino acids can also be used toreplace the amino acid in the tandem decapeptide sequence. Preferablyonly conservative replacements are carried out. Conservativereplacements are amino acid substitutions in which bulky amino acids arereplaced by bulky amino acids, aromatic amino acids by aromatic aminoacids, etc. These concepts are well known to those skilled in the art.

[0041] Spacers can be placed between the peptides according to theinvention. This allows for the formation of multimers. Suitable spacersare known in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042]FIG. 1: Competition for binding of iodinated GnRH with antiserumby GnRH (open circles), GnRH-II (solid circles), control peptide (solidsquares) and no peptide (asterix). Serum is diluted 1/10000 andincreasing peptide concentrations (0.25, 2.5 25 pmol) per well wereadded. In graphs A-C, sera with increasing binding capacity for GnRH-IIare shown. Horizontal axis: amount of peptide (pmol per well); verticalaxis: Binding capacity (counts per minute).

[0043]FIG. 2: Percentage of maximum binding of antiserum to iodinatedGnRH after competition with GnRH-I (dark bars) or GnRH-II (light bars)for sera obtained after immunization with the peptides indicated in theleft top corner of each graph. Antiserum obtained 3 weeks after boosterimmunization was diluted 1:10000 and peptides for competition were addedin 250 nmol per liter. Horizontal axis: animal identification #;Vertical axis: percentage of maximum binding.

[0044]FIG. 3: Percentage of maximum binding of antiserum to iodinatedGnRH after competition with GnRH-I (dark bars) or GnRH-II (light bars)for antiserum against pE1A collected at 7 wpv (A) and at 10 wpv (B) andfor antiserum against S4A collected at 7 wpv (C) and at 10 wpv (D).Antiserum obtained at 7 wpv and at 10 wpv was tested in an 1/2000 and1/10000 dilution respectively. 25 pmol peptide for competition was addedper well. Horizontal axis: animal identification #; Vertical axis:percentage of maximum binding.

[0045]FIG. 4: Immunocastration efficacy score of several groups of pigsimmunized with GnRH-tandem-dimer peptides (62 mg) conjugated toovalbumin and Specol used as an adjuvant. The staring peptide was GnRHtandem (Cys-OH) dimer (abbreviated Cys-OH). Of this peptide all alaninescan peptides were used for immunization. Immunocastration efficacy wasrated on a scale of 1 to 4 (1=immunocastration did not occur, 2=aminority of the pigs was immunocastrated, 3=a majority of the pigs wasimmunocastrated, 4 all pigs were immunocastrated).

DETAILED DESCRIPTION OF THE INVENTION

[0046] In a feature of the invention, the peptides according to theinvention have the general formula given by (SEQ ID NO: 4), or by thefollowing one-letter code:

pEHWSYkLRPGQHWSYkLRPGC#

[0047] In this general formula, Q stands for Gln and may be preceded byX where X stands for a spacer. Several of these amino acids have beenreplaced with other amino acids. In the formulas the position of thesubstitution is depicted bold and underscored. Capital letters stand forLevorotatory amino acids, lower case letters stand for Dextrorotatoryamino acids, e.g. K: L-Lys; k:D-Lys. Subsequently, their immunogenicresponse has been determined when coupled to a carrier, generallyovalbumin, but other carriers such as KLH, BSA can be used.

[0048] It will be clear that the sequence of the GnRH-II peptide (SEQ IDNO: 2) determines which of the amino acids can be replaced in suchmanner that an effective discrimination based on the immune response isstill possible between the two sequences GnRH-I, and GnRH-II.

[0049] To determine whether antibodies raised by the GnRH vaccinesaccording to the present invention discriminated between GnRH-I andGnRH-II, a GnRH antibody binding assay was performed in order todetermine whether antibodies rose against GnRH-1-tandem dimer peptide orits alanine replacement analogs bind to GnRH-II or lack binding toGnRH-II. Serum dilutions were preincubated with either GnRH-I, GnRH-II,a control peptide, or no peptide. Next, iodinated GnRH-I was added tocompete with the preincubated peptides for binding to the antibodies.

[0050] This procedure was performed for serum collected before and afterbooster immunization, as specificity of the antibodies may increaseafter booster immunization.

[0051] When the peptides according to the invention were used as theconjugate with ovalbumin (OVA-conjugate) and compared to controls, allshowed effectiveness in the immunocastration of young male pigs.Comparison with the known G6k-GnRH tandem dimer OVA conjugate (seeTable 1) showed that the peptides according to the invention presentedcomparable or similar effectiveness even though their resemblance to theself-hormone GnRH-I has decreased. The peptides according to theinvention give an immunogenic response that allows for the effectivediscrimination between GnRH-I and GnRH-II. These peptides resulted insmall testes and low testosterone levels. More specifically, thepeptides expressing a low testes weight and a low testosterone level areR8A, G10A and S4A. The preferred peptides based on immunologicalselectivity between GnRHI and GnRH II are S4A and pE1A.

[0052] In a preferred embodiment, the peptide is selected from the groupconsisting of pEHWAYkLRPGQHWAYkLRPGC# (SEQ ID NO: 5),pEHWSYkLAPGQHWSYkLAPGC# (SEQ ID NO: 6) and pEHWSYkLRPAQHWSYkLRPAC# (SEQID NO: 7). It is more preferably selected from the group consisting ofpEHWSYkLAPGQHWSYkLAPGC# (SEQ ID NO: 6); and pEHWSYkLRPAQHWSYkLRPAC# (SEQID NO: 7).

[0053] In the peptides according to the invention, dimerization of thetandem units can, for example, take place via the carboxyl-terminus orvia the amino-terminus. Two tandem units may, for instance, be dimerizedby means of a disulfide or thioether bridge. To dimerize the tandemsequences, the Cys at position 21 can be used, or Cys can be synthesizedbefore the glutamic acid at position 1. Other methods to dimerize ormultimerize the GnRH-tandem units can also be found in the prior art. Ifthe Cys at position 21 is involved in the dimerization and accordinglynot available for coupling, it is likewise possible to use another aminoacid of the tandem that can be coupled. If the dimerization ormultimerization results in the loss of accessible sites where a carriercompound can be conjugated, it is sufficient to restrict the choice ofreplacement amino acids to an amino acid with an appropriate side chain.Such a replacing amino acid can be, for example, L or D-Lys, L or D-Gluor another amino acid containing a side chain that allows coupling to acarrier compound. Both L- and D-substitutions have been tested and foundto have the same effect.

[0054] More in particular, an example of such a preferred peptideaccording to the invention is a D-Lys⁶-tandem-GnRH dimer (SEQ ID NO: 8)according to the following formula: 1                    21#EHWSYkLRPGQHWSYkLRPGC                      | #EHWSYkLRPGQHWSYkLRPGC22                   42

[0055] In this example of an embodiment of the invention, it is possibleto replace one of the amino acids of the tandem-dimer by another aminoacid.

[0056] Other peptides or peptide sequences or coupled peptide sequencesin which monomerized, dimerized or multimerized GnRH tandem units arepresent that contain amino acid substitutions are also part of theinvention.

[0057] The invention further provides a composition which comprises apeptide brought into an immunogenic form. As a skilled worker knows,there are different methods of producing an immunogenic form of asubstance that is, in itself, not immunogenic. One possibility is tocouple the peptide of the invention to a suitable carrier protein. Asuitable carrier protein is ovalbumin, KLH or BSA. In a tandem peptide,a cysteine at the - or C-terminus can be suitably used for a chemicalcoupling. In the tandem-dimer peptide, coupling can also be performedusing the plain or the modified side chain of (D-) lysine, (D-)glutamine, or any other modified amino acid replacing amino acids of thepeptide sequence. Suitable coupling methods and carrier proteins arewell known to those of ordinary skill in the art.

[0058] According to the invention, there is preferred a compositionwhich is characterized in that it comprises an immunogenic conjugate ofa protein, such as ovalbumin, and a peptide or peptide composition.

[0059] A composition according to the invention can be used in the formof a vaccine. To this end, the composition can be produced in a formthat is suitable for administration. By administration of a vaccineaccording to the invention, an immunogenic response against GnRH isgenerated, preferably an immunogenic response against GnRH-I.

[0060] The invention therefore also provides a method of immunizing amammal against GnRH-I through vaccination of the mammal with a vaccineaccording to the invention. In a preferred embodiment, the inventionprovides a method of selectively immunizing a mammal against GnRH-I witha vaccine according to the invention.

[0061] Of course, the vaccine preparation according to the invention canbe combined with at least one immunoadjuvant. Suitable immunoadjuvantsare known to those skilled in the art. A preferred adjuvant according tothe invention can be Specol or a double oil emulsion, but otheradjuvants that elicit no or only mild side-reactions can be used aswell. The invention can be used in methods for immunizing individualsselected from a wide range of vertebrates, and particularly mammals,against GnRH-I. In a preferred embodiment of the invention, the vaccinecan be administered in a single dose, which has the same efficacy as thepresently known vaccines which must be administered in two-dose form.Immunization against GnRH-I, preferably selective, could, for instance,be used for the sterilization of, e.g., small domestic animals such asmale and female cats and dogs, or for the treatment of aggressiveness inmale dogs and bulls. Other conceivable reasons of immunization againstGnRH-I with the present invention are preventing heat in female animalssuch as dogs, cats and cows, and preventing or treating restlessness inmale animals being fattened for slaughter. In human health care,immunization against GnRH, preferably selective against either GnRH-I orGnRH-II, can be used in the treatment of prostate cancer and breastcancer and, if required, in the treatment of some forms of pituitarycarcinoma.

[0062] A preferred embodiment is a method of improving the carcassquality of pigs, wherein the pigs are vaccinated with such a vaccinepreparation according the invention. The invention is illustrated in thefollowing experimental part.

[0063] I. Immunocastration of Pigs

[0064] Materials and Methods

[0065] Materials

[0066] Acetonitrile (ACN) was HPLC-S gradient grade, N-methylpyrrolidone(NMP), diisopropylethylamine (DIEA) dimethylformamide (DMF),trifluoroacetic acid (TFA) and piperidine were peptide synthesis gradeand were all obtained from Biosolve (Valkenswaard, NL).N-hydroxybenzotriazole (HOBt) and2-(1H-benzotriazol-1-yl-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) were obtained from Richelieu Biotechnologies Inc. (Hamon,Canada). Benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate (PyBOP) was obtained from Novabiochem (Laufelfingen,Switzerland). Thioanisole (TA), ethanedithiol (EDT), dimethylsulfoxide(DMSO), pentane and dimethylaminopyridine (DMAP) were pro-analysis gradeand were obtained from Merck (Darmstad, Germany). Diethylether waspurified over a column of activated, basic aluminum oxide before use.Amino acid derivatives and resins were obtained from BachemFeinchemicalien AG (Bubendorf, Switzerland).

[0067] Multiple Peptide Synthesis (MPS)

[0068] A Hamilton Microlab 2200 was programmed to deliver washingsolvents and reagents to a rack with 40 individual 4 ml columns withfilter, containing 30 mmol of resin for peptide synthesis. The columnswere drained after each step by vacuum. The coupling cycle was based onFmoc chemistry using double coupling steps:

[0069] 1. NMP wash (1 ml)

[0070] 2. 30% (v/v) piperidine/NMP (3 min, 0.5 ml)

[0071] 3. 30% (v/v) piperidine/NMP (17 min, 0.5 ml)

[0072] 4. NMP wash (5×1 ml)

[0073] 5. double coupling (2×30 min)

[0074] 6. NMP wash (2×1 ml)

[0075] Coupling step: Fmoc-amino acid in NMP (0.4 M, 0.25 ml), HBTU/HOBt(0.45 M, 0.22 ml) in DMF, and DIEA (2 M, 0.2 ml) in NMP were transferredto the reaction vessel and allowed to react for 30-50 min. The reactionmixture was drained and the coupling procedure was repeated once.

[0076] After coupling of the last amino acid, the Fmoc group was cleavedwith 30% piperidine/NMP, the peptides were washed, acetylated for 30min. using NMP/acetic anhydride/DIEA 10/1/0.2, washed again, and dried.The peptides were deprotected and cleaved for 2 h in a mixture of 1.5 mlof TFA/phenol/TA/water/EDT 10/0.75/0.5/0.5/0.25 (reagent K). Thecleavage mixture was filtered, the resin was washed with 0.5 ml TFA, andthe peptide was precipitated by adding 13 ml pentane/diethylether 1/1.After centrifugation, the precipitate was extracted again withpentane/diethylether. The precipitate was dried, dissolved in ACN/water1/1 and lyophilized. This procedure yields, depending on molecularweight, 25 to 70 mg of peptide.

[0077] Peptide sequences synthesized in single letter amino acid codeare summarized in Table 1. TABLE 1 Amino acid sequence in single lettercode of peptides synthesized peptide amino acid sequence G6k-GnRH-tandempEHWSYkLRPGQHWSYkLRPGC# (SEQ ID NO: 9) pE1A-G6k-GnRH-tandem * AHWSYkLRPGAHWSYkLRPGC# (SEQ ID NO: 10) S4A-G6k-GnRH-tandem pEHW AYkLRPGQHWAYkLRPGC# (SEQ ID NO: 5) Y5A-G6k-GnRH-tandem pEHWS AkLRPGQHWS AkLRPGC#(SEQ ID NO: 11) L7A-G6k-GnRH-tandem pEHWSYk ARPGQHWSYk ARPGC# (SEQ IDNO: 12) R8A-G6k-GnRH-tandem pEHWSYkL APGQHWSYkL APGC# (SEQ ID NO: 6)P9A-G6k-GnRH-tandem pEHWSYkLR AGQHWSYkLR AGC# (SEQ ID NO: 13)G10A-G6k-GnRH-tandem pEHWSYkLRP AQHWSYkLRP AC# (SEQ ID NO: 7)

[0078] Analytical HPLC

[0079] For analysis of peptides, we used an LC-MS (electrospray) system,which consists of two Waters pumps model 510, a Waters gradientcontroller model 680, a Waters WISP 712 auto-injector, and a Waters 991photodiode array detector. The mass spectrometer was a Micromass QuattroII sq, which was used in positive ion mode. Products were analyzed in alinear gradient from 10% ACN/water with 0.05% TFA to 70% ACN/water with0.05% TFA for 30 min on a Waters Delta Pak C18-100 Å (3.9×150 mm, 5 mm)column at 1 ml/min at 215 nm. All products were between 40-70% pureaccording to the peak area.

[0080] Dimerization

[0081] Crude products were dimerized by dissolving the products in 20%DMSO in water. The pH was adjusted to 5-6 with 1% NH₄HCO₃, maintaining aclear solution. Correction of pH was done with 1% acetic acid. Afterstirring at room temperature for at least 5 h, the products were storedat −20° C. until purification.

[0082] Preparative HPLC

[0083] Peptide purifications were carried out using a Waters Prep 4000liquid chromatograph, equipped with a Waters RCM module with twoPREPPAK™ cartridges plus guard cartridge (40×210 mm or 25×210 mm) filledwith delta-Pak C18-100 Å (15 mm) material. In general, purificationswere run using the same eluents as in analytical HPLC, but at a gradientspeed of 0.5% ACN/min and a flow rate of 40 or 100 ml/min. Peptides weredetected at 215-230 nm using a Waters 486 spectrophotometer with apreparative cell. The peptides were lyophilized and purity wasdetermined to be at least 90%.

[0084] Conjugate Preparation

[0085] For conjugation viaN-ethyl-N=-(3′-dimethylaminopropyl)carbodiimide hydrochloride (“EDC”) tochicken egg albumin (“OVA”), an equal weight of both the peptide and thecarrier protein were dissolved separately in milli Q water and bothsolutions were mixed well. Next, a 10-fold excess, based on weightequivalents, of EDC was dissolved in milli Q water. Subsequently, thissolution was slowly added to the solution of peptide/OVA undercontinuous stirring; pH of this final solution is 5. After at least 6 hslowly shaking, the product was dialyzed (MW cut-off 10,000) against a300 times excess of milli Q water for two days. Water was refreshedtwice a day. The loading was calculated from comparative amino acidanalysis of the conjugate and the carrier protein. Amino acid analysiswas performed using a Waters Pico-Tag system, after hydrolysis in aPico-Tag workstation using 6N HCl at 150° C. for 1 h, and derivatizationwith phenyl isothiocyanate.

[0086] According to the amino acid analysis, the conjugates containedbetween 0.3 and 0.5 mg of peptide per mg of carrier protein, withexception of the P9A-G6k-GnRH-tandem-dimer conjugate which onlycontained 0.16 mg peptide per mg ovalbumin.

[0087] The G6k-GnRH-tandem-dimer OVA conjugate is abbreviated by G6k-TD.The conjugates with alanine substitutions are abbreviated by the aminoacid in the native GnRH sequence that is replaced, its position and an Afor the Alanine replacement. For example, pE1A-G6k-GnRH-tandem-dimer OVAconjugate is pE1A.

[0088] Emulsion Preparation

[0089] Specol (Special Oil Phase, ID-DLO, Lelystad, The Netherlands)consisting of two detergents in a light mineral oil was used as oilphase (Bokhout et al., 1981). The water-in-oil-(WIO)-emulsions wereprepared using an Ultra Turrax (Janke and Kunkel, Staufen, Germany) witha stirring bar i 25 mm. The oil phase Specol (5 parts v/v) was broughtinto a 25 ml glass vessel and the water phase (4 parts v/v) consistingof the conjugate in milli Q water was slowly added while the emulsionwas stirred. After the water phase was added, the emulsion was stirredfor half a minute at the same rotation speed (15000 rpm). Emulsions werestored overnight at 4° C. to check stability and were administered tothe animals the next day.

[0090] Animals

[0091] Male piglets, approximately 10 weeks of age, were involved inthis experiment. The crossbred piglets were housed in half-slatted pensand were given ad libitum access to feed and water.

[0092] Immunization

[0093] The piglets were randomly assigned to the treatments, 6 or 7piglets per treatment. All animals were injected with 2 ml emulsioncontaining the dimerized tandem GnRH conjugates (i.e., 62 mg peptide) oran emulsion without antigen. Injections were administeredintramuscularly in the neck at the start of the experiment (day 0) and 7weeks later (7 wpv). Thirteen weeks after initial immunization (13 wpv)all animals were slaughtered.

[0094] Measurements and Blood Sampling

[0095] Animals were weighed at day 0 and 7 and 13 wpv. Testis sizes weredetermined by measuring testis length with Vernier calipers at day 0,and 7, 10 and 13 weeks thereafter. Testis sizes were recorded as averageof both testicles.

[0096] Blood samples were taken via puncture of the vena jugularis onthe same days testis sizes were measured, and also 4 weeks after theinitial immunization. Blood samples were kept overnight at 4° C. and thenext day serum was obtained by centrifugation (1500 g, 15 min). Serumsamples were stored at −20° C. until assayed.

[0097] Evaluation After Slaughter

[0098] After slaughter testes were removed, dissected free of epididymesand weighed. Testes weights were recorded as average of both testicles.

[0099] Peptide Antibodies

[0100] Antibodies to the peptides used for immunization were determinedwith an ELISA. Peptides were coated in the wells of a microtitreplateusing glutardialdehyde (GDA). GDA was coated to the surface of the wellsby incubation with 0.2% GDA in 0.1 M phosphate buffer (pH 5) for 4 hoursat room temperature. Plates were rinsed 3 times for 10 minutes with 0.1M phosphate buffer (pH 8). One microgram peptide in 100 ml phosphatebuffer (0.1 M, pH 8) was coated per well by incubating for 3 hours at37° C. Plates were stored at −20° C. until used. Thawed plates wererinsed 3 times for 10 minutes with milli Q water containing 8.2 g NaCl,1.15 g Na₂HPO₄.2H₂O, 0.20 g NaH₂PO₄.2H₂O and 5 ml of a 10% Tween 80solution in water per litre water.

[0101] Serial serum dilutions of the antipeptide sera were allowed toreact with the coated peptides for 1 hour at 25° C. After rinsing 3times for 10 minutes, goat-anti-pig IgG coupled to horseradishperoxidase (Dako, Glastrup, Denmark) was introduced as second antibodyfor 1 hour and ABTS (Boehringer, Mannheim, Germany) ((250 ml (2 g/100ml) in 10 ml substrate buffer to which 20 ml H₂O₂ (3% solution)) wasused as substrate. Absorption was measured at 405 nm.

[0102] GnRH Antibodies

[0103] Antibodies to GnRH were determined as described by Meloen et al.(Vaccine 12, 741-746 (1994)). Serial dilutions of the pig antisera wereallowed to bind to ¹²⁵I-GnRH. Titers are expressed as percentage bindingof ¹²⁵I-GnRH at a given serum dilution.

[0104] Testosterone

[0105] Testosterone levels in serum were determined using a Coat-a-Countkit purchased from DPC laboratories, Los Angeles, Calif.

[0106] Results

[0107] Testis Size and Testis Weight

[0108] Seven weeks after the first immunization, immunocastrationeffects already could be observed by measuring testicle sizes. Threetreatments (R8A, G10A and G6k-TD) showed hardly any increment (<10 mm)of average testis size at time of booster. These treatments weresuccessful with testis weights at slaughter being 70 grams or less.Other treatments that were effective in terms of low testes weights arepE1A and S4A, while in groups Y5A, L7A and P9A, 2, 1 and 1 animals didnot respond to the immunization respectively (Table 2). Individualtestis weight of immunocastrated animals did not exceed 70 grams,resulting in a clear difference between immunocastrated and notimmunocastrated animals. TABLE 2 Effectiveness of the differenttreatments according testis weight (g) number testis weight (g)responders/ treatment individual testis weight(g) (median (range)) totalnumber G6k-TD 10, 10, 10, 12, 16, 70 11 (10-70) 6/6 pE1A 16, 18, 33, 36,53, 55, 65 36 (16-65) 7/7 S4A 10, 14, 15, 36, 40, 43, 69 36 (10-69) 7/7Y5A 15, 19, 34, 44, 69, 210, 235  44 (15-235) 5/7 L7A 12, 13, 15, 20,24, 29, 195  20 (12-195) 6/7 R8A 10, 10, 12, 15, 17, 19, 19 15 (10-19)7/7 P9A 8, 9, 20, 23, 31, 57, 300 23 (8-300) 6/7 G10A 11, 13, 14, 14,15, 30, 39 14 (11-39) 7/7 Controls 150, 173, 204, 206, 236  204(150-236) 0/5

[0109] Antibody Response

[0110] Mean antibody titers against the peptides used for immunizationare given in table 3. Mean antibody titers of pigs treated with H2A andP9A are lower than peptide antibody titers of other treatments.

[0111] Antibody titers of individual animals against the differentpeptides ranged from 2 to 4. Within a treatment, animals that were notimmunocastrated showed the lowest antipeptide titer. Animals treatedwith H2A and W3A were not immunocastrated, but significant antipeptidetiters were present.

[0112] GnRH antibody binding percentages at 1/2000 serum dilution wereundetectable or low in H2A and W3A groups. However, at a 1/200 serumdilution, antibodies were detectable in sera of all animals of group H2Aand three animals of group W3A.

[0113] Animals with low average GnRH antibody titers were notimmunocastrated. High antibody titers did always result in successfullycastrated animals. Testicle weight of animals with intermediate antibodytiters varied from 15 to 300 gram.

[0114] Average GnRH antibody titers per treatment showed a clearrelation with testis weight (median) per treatment (Table 3).

[0115] Testosterone

[0116] Testosterone levels of all successfully treated animals were lowand decreased after the second immunization. However, a majority (n=31)of the animals reflected a castration effect as early as four weeksafter initial immunization by showing undetectable testosterone levels.Testis weight of these animals varied from 8-36 grams.

[0117] The majority of the animals were thus effectively immunized afterthe administration of a single dose.

[0118] Although testosterone levels at slaughter were low for allimmunocastrated animals, two animals with testes weights of 65 and 70grams had significant serum testosterone levels of 3.80 and 1.18 pmol/mlrespectively.

[0119] The peptides that resulted in low testes weight combined withundetectable testosterone levels are considered the most effectivepeptides for the immunocastration of pigs. These peptides are S4A, R8Aand G10A.

[0120] Testosterone levels at slaughter of pigs that were notimmunocastrated animals varied between 0.46 and 48.91 pmol/ml. TABLE 3Effect of different treatments on mean testis weight, peptide antibodytiter, GnRH antibody binding percentage and LH and testosterone levelsin serum. anti testosterone peptide (pmol/ml) testis weight titer atGnRH binding at 13 wpv Treatment (median range) 10 wpv (average) median(range) G6k-TD 11 (10-70) 3.32 17.2 n.d. (n.d.-1.18) pE1A 36 (16-65)3.24 15.7 n.d. (n.d.-3.80) S4A 36 (10-69) 3.19 16.9 n.d. (n.d.) Y5A  44(15-235) 2.92 11.3 n.d. (n.d.-9.44) L7A  20 (12-195) 3.52 17.6 n.d.(n.d.-48.91) R8A 15 (10-19) 3.34 17.7 n.d. (n.d.) P9A 23 (8-300) 2.6215.8 n.d. (n.d-11.57) G10A 14 (11-39) 3.19 17.6 n.d. (n.d.)

[0121] II. Replacement of D-Amino Acid with L-Amino Acid

[0122] D-lysine (k) at positions 6, 16, 27, and 37 in a tandem dimer isreplaced by an L-lysine (K) and the resulting peptide tested (Table 4).

[0123] G6k-GnRH-tandem: pEHWSYkLRPGQHWSYkLRPGC# (SEQ ID NO: 9)

[0124] G6K-GnRH-tandem: pEHWSYKLRPGQHWSYKLRPGC# (SEQ ID NO: 14) TABLE 4testosterone number in pmol/ml, testis weight (g) responders/ antipeptide GnRH-binding at 13 wpv, treatment median (range) total titer at10 wpv (average) (median) G6k-TD 11 (10-70) 6/6 3.32 17.2 n.d. G6K-TD 21(9-175) 5/6 2.70 15.6 n.d. Controls  204 (150-236) 0/5 n.d. n.d. 2.05

[0125] Replacement of D-lysine by L-lysine does not alter efficacy ofthe vaccine antigen.

[0126] III. Discrimination Between GnRh-I and GnRH-II

[0127] A GnRH antibody binding competitive radioimmunoassay wasperformed in order to determine whether antibodies raised againstG6k-GnRH-tandem (SEQ ID NO: 9) dimer peptide or its alanine replacementanalogs bind to GnRH-II or lack binding to GnRH-II. Serum dilutions werepreincubated with either GnRH-I, GnRH-II, a control peptide or nopeptide. Next, iodinated GnRH-I was added to compete with thepreincubated peptides for binding to the antibodies.

[0128] This procedure was performed for serum collected before and afterbooster immunization, as specificity of the antibodies may increaseafter booster immunization due to antibody maturation.

[0129] Materials and Methods

[0130] Serum samples of the 10 wpv bleeding (3 weeks post boosterimmunization) were diluted 1/10000 in PBS with 0.4% BSA (dilutionbuffer). Fifty μl serum dilution was put in microwell plates and 25 μlpeptide solution (0, 0.25, 2.5 or 25 pmol peptide in dilution buffer perwell) was added. This mixture was allowed to incubate for 24 hours at 4°C. The next day, 25 μl iodinated GnRH (approximately 13000 cpm) wasadded and after overnight storage (4° C.) unbound peptide was separatedfrom bound peptide with charcoal. After centrifugation, supernatant wasseparated, counted and the percentage of iodinated GnRH bound to theantibodies was calculated.

[0131] Antibodies collected after the first immunization (7 wpv) weretested in a 1/2000 serum dilution and only one peptide dilution wastested (25 pmol/well).

[0132] Results

[0133] Binding of iodinated GnRH at a 1/10000 serum dilution wasobserved for the sera of all treatments except for H2A and W3A sera.Competition with GnRH-I resulted in a dramatic reduction of binding ofiodinated GnRH to the antiserum (see FIG. 1). At the sera dilution wetested, inhibition of binding of iodinated GnRH by GnRH-I was dosedependent for most sera. As expected, none of the antisera bound to thecontrol peptide.

[0134] Competition by GnRH-II for antibody binding to iodinated GnRHresulted in a reduced binding in all sera of R8A and G10A treatments, inmost of the sera of treatments P9A and TDK and in half of the sera oftreatments Y5A and L7A (see FIG. 2). However, antibodies ofsubstantially all animals of the pE1A and the S4A group completelylacked binding to GnRH-II, and were thus specific for GnRH-I.

[0135] The antibody binding results obtained with the prebooster serawere different from the 10 wpv sera. Antisera of the pE1A and S4Atreatments have been tested for their ability to recognize GnRH-I orGnRH-II (see FIG. 3). For both peptides, 10 wpv sera from 6 out of 7animals did not bind to GnRH-II. Of the pE1A sera obtained beforebooster immunization, 4 out of 7 animals showed binding to GnRH-II. Twosera did not recognize GnRH-II and one serum showed no binding toiodinated GnRH. The results of the S4A prebooster sera are contradictoryto the results of the 10 wpv sera. The prebooster sera of all animals ofthe S4A group did recognize GnRH-II, with the inhibition of bindingcapacity of iodinated GnRH being similar for both GnRH-I and GnRH-II.

1 13 1 10 PRT Sus scrofa MISC_FEATURE (1)..(1) X=pyroglutamic acid 1 XaaHis Trp Ser Tyr Gly Leu Arg Pro Xaa 1 5 10 2 10 PRT Homo sapiensMISC_FEATURE (1)..(1) X=pyroglutamic acid 2 Xaa His Trp Ser His Gly TrpTyr Pro Xaa 1 5 10 3 20 PRT Artificial Sequence A peptide suitable foreliciting an immune response against forms GnRH/ LHRH. 3 Xaa His Xaa SerTyr Gly Leu Arg Pro Gly Gln His Xaa Ser Tyr Gly 1 5 10 15 Leu Arg ProXaa 20 4 21 PRT Artificial Sequence A peptide suitable for eliciting animmune response against forms GnRH/ LHRH. 4 Xaa His Trp Ser Tyr Xaa LeuArg Pro Gly Xaa His Trp Ser Tyr Xaa 1 5 10 15 Leu Arg Pro Gly Xaa 20 521 PRT Artificial Sequence A peptide suitable for eliciting an immuneresponse against forms GnRH/ LHRH. 5 Xaa His Trp Ala Tyr Xaa Leu Arg ProGly Xaa His Trp Ala Tyr Xaa 1 5 10 15 Leu Arg Pro Gly Xaa 20 6 21 PRTArtificial Sequence A peptide suitable for eliciting an immune responseagainst forms GnRH/ LHRH. 6 Xaa His Trp Ser Tyr Xaa Leu Ala Pro Gly XaaHis Trp Ser Tyr Xaa 1 5 10 15 Leu Ala Pro Gly Xaa 20 7 21 PRT ArtificialSequence A peptide suitable for eliciting an immune response againstforms GnRH/ LHRH. 7 Xaa His Trp Ser Tyr Xaa Leu Arg Pro Ala Xaa His TrpSer Tyr Xaa 1 5 10 15 Leu Arg Pro Ala Xaa 20 8 42 PRT ArtificialSequence A peptide suitable for eliciting an immune response againstforms GnRH/ LHRH. 8 Xaa His Trp Ser Tyr Xaa Leu Arg Pro Gly Xaa His TrpSer Tyr Xaa 1 5 10 15 Leu Arg Pro Gly Cys Xaa His Trp Ser Tyr Xaa LeuArg Pro Gly Xaa 20 25 30 His Trp Ser Tyr Xaa Leu Arg Pro Gly Cys 35 40 921 PRT Artificial Sequence A peptide suitable for eliciting an immuneresponse against forms GnRH/ LHRH. 9 Xaa His Trp Ser Tyr Xaa Leu Arg ProGly Xaa His Trp Ser Tyr Xaa 1 5 10 15 Leu Arg Pro Gly Xaa 20 10 21 PRTArtificial Sequence A peptide suitable for eliciting an immune responseagainst forms GnRH/ LHRH. 10 Xaa His Trp Ser Tyr Xaa Leu Arg Pro Gly AlaHis Trp Ser Tyr Xaa 1 5 10 15 Leu Arg Pro Gly Xaa 20 11 21 PRTArtificial Sequence A peptide suitable for eliciting an immune responseagainst forms GnRH/ LHRH. 11 Xaa His Trp Ser Ala Xaa Leu Arg Pro Gly XaaHis Trp Ser Ala Xaa 1 5 10 15 Leu Arg Pro Gly Xaa 20 12 21 PRTArtificial Sequence A peptide suitable for eliciting an immune responseagainst forms GnRH/ LHRH. 12 Xaa His Trp Ser Tyr Xaa Ala Arg Pro Gly XaaHis Trp Ser Tyr Xaa 1 5 10 15 Ala Arg Pro Gly Xaa 20 13 21 PRTArtificial Sequence A peptide suitable for eliciting an immune responseagainst forms GnRH/ LHRH. 13 Xaa His Trp Ser Tyr Xaa Leu Arg Ala Gly XaaHis Trp Ser Tyr Xaa 1 5 10 15 Leu Arg Ala Gly Xaa 20

What is claimed is:
 1. A peptide comprising a modified tandem GnRHdecapeptide sequence capable of inducing an immunogenic response thatallows for discrimination between different types of GnRH.
 2. A peptidecomprising a modified tandem GnRH decapeptide sequence which allows fora testosterone level that is essentially nondetectable after vaccinationwith the peptide in a suitable dosage.
 3. The peptide of claim 1,comprising at least two coupled GnRH decapeptide sequences, wherein atleast one amino acid of said coupled GnRH decapeptide sequences isreplaced by a different amino acid.
 4. The peptide of claim 3, whereinthe different amino acid is Ala.
 5. The peptide of claim 1, wherein thepeptide is selected from the group consisting of SEQ ID NO: 5, SEQ IDNO: 6, and SEQ ID NO:
 7. 6. The peptide of claim 1, that is dimerized.7. The peptide of claim 6, conjugated with a carrier compound.
 8. Thepeptide of claim 7, wherein the carrier compound is a protein.
 9. Thepeptide of claim 8, wherein the carrier compound is ovalbumin.
 10. Avaccine comprising a peptide in accordance with claim
 1. 11. The vaccineof claim 10, further comprising an adjuvant.
 12. The vaccine of claim11, wherein the adjuvant is an oil phase of an emulsion selected from agroup consisting of a water-in-oil emulsion and a double oil emulsion.13. A method for vaccinating a mammal against GnRH-I with the vaccine ofclaim
 10. 14. The method according to claim 13, wherein the vaccine isadministered in a single dose.
 15. The vaccine of claim 10 that issufficiently active for administration in a single dose for theessential immunocastration of pigs.
 16. A method to affect one or morereproductive or behavioral characteristics of a mammal, wherein saidmammal is vaccinated in accordance with the method according to claim13.
 17. A method for immunizing a mammal against GnRH, comprisingvaccinating the mammal with the vaccine of claim
 16. 18. A method ofimmunocastrating a pig, comprising vaccinating the pig in accordancewith the method of claim
 17. 19. Antibodies against GnRH-II obtainableby a method comprising a step wherein an immune response is elicited tothe peptide of claim
 1. 20. A vaccine against GnRH-II comprising thepeptide of claim
 1. 21. A composition for the treatment of prostatecancer comprising the peptide of claim
 1. 22. A method for the treatmentof prostate cancer comprising administration of a suitable dose of acomposition comprising a peptide that elicits at least an immunogenicresponse against GnRH-II.
 23. The method according to claim 13, whereinthe vaccine is a selective vaccine for vaccination against GnRH-I. 24.The peptide of claim 3, wherein said different amino acid is Ala. 25.The peptide of claim 3, wherein said peptide is selected from a groupconsisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO:
 7. 26. Thepeptide of claim 4, wherein said peptide is selected from a groupconsisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO:
 7. 27. Thepeptide of claim 1, wherein said peptide is multimerized.
 28. Thepeptide of claim 3 wherein said peptide is dimerized.
 29. The peptide ofclaim 3 wherein said peptide is multimerized.
 30. The peptide of claim 4wherein said peptide is dimerized.
 31. The peptide of claim 5 whereinsaid peptide is dimerized.
 32. A vaccine comprising the peptide ofclaim
 1. 33. A vaccine comprising the peptide of claim
 3. 34. A vaccinecomprising the peptide of claim
 5. 35. A vaccine comprising the peptideof claim
 6. 36. A vaccine comprising the peptide of claim
 7. 37. Avaccine comprising the peptide of claim
 9. 38. A method for vaccinatinga mammal against GnRH-I, said method comprising vaccinating the mammalwith the vaccine of claim
 11. 39. A method for vaccinating a mammalagainst GnRH-I, said method comprising vaccinating the mammal with thevaccine of claim
 12. 40. The vaccine of claim 11 that is sufficientlyactive for administration in a single dose for the essentialimmunocastration of pigs.
 41. The vaccine of claim 12 that issufficiently active for administration in a single dose for theessential immunocastration of pigs.
 42. A method to affect one or morereproductive or behavioral characteristics of a mammal, wherein saidmammal is vaccinated in accordance with the method according to claim14.
 43. A method to affect one or more reproductive or behavioralcharacteristics of a mammal, wherein said mammal is vaccinated inaccordance with the method according to claim
 15. 44. Antibodies againstGnRH-II obtainable by a method comprising a step wherein an immuneresponse is elicited to the peptide of claim
 3. 45. Antibodies againstGnRH-II obtainable by a method comprising a step wherein an immuneresponse is elicited to the peptide of claim
 5. 46. Antibodies againstGnRH-II obtainable by a method comprising a step wherein an immuneresponse is elicited to the peptide of claim
 6. 47. Antibodies againstGnRH-II obtainable by a method comprising a step wherein an immuneresponse is elicited to the peptide of claim
 7. 48. Antibodies againstGnRH-II obtainable by a method comprising a step wherein an immuneresponse is elicited to the peptide of claim
 9. 49. A vaccine againstGnRH-II, said vaccine comprising the peptide of claim
 5. 50. Acomposition for treating prostate cancer, said composition comprisingthe peptide of claim 5.